KR20040058194A - Storage-stable fibrinogen solutions - Google Patents

Abstract

The method of the present invention provides a ready-to-use, biologically suitable mammalian fibrinogen stable storage method that remains fluid even at high concentrations and can be quickly and easily used for a long time for tissue binder preparation. Also provided are sterile, storage-stable, water-soluble fibrinogen products prepared through the process of the present invention, wherein the fibrinogen is maintained in a liquid form that is readily available for a long time and lacks an active agent such as spontaneous clotting (ie, thrombin / Ca ^++). There is no lower clot formed, and biological activity (i.e. the ability to rapidly form fibrin clot when exposed to thrombin and Ca ⁺⁺ and mixed vigorously) is maintained.

Description

Storage-stable fibrinogen solutions

Fibrinogen is a plasma protein that plays an important role in the final stage of coagulation in mammals to maintain hemostasis and prevent blood loss. In mammals, clot formation, ie, blood coagulation, in the final step, the fibrinogen in monomeric form reacts with thrombin and activated factor XIII in the presence of calcium ions to form a fibrin clot comprising a cross-linked fibrin polymer. This occurs through a complex series of stepwise reactions.

The fibrinogen monomer, present at 2-4 grams / liter of plasma protein, consists of three pairs of bisulfide-linked polypeptide chains. This is represented by (Aα) ₂ , (Bβ) ₂ , which represents two small amino terminal peptides of α and β chains (respectively) and γ ₂ . Cleavage of fibrinopeptide A from fibrinogen by thrombin results in compound, fibrin I, followed by cleavage of fibrinopeptide B resulting in the final fibrin II compound. The cleavage only slightly reduces the molecular weight of fibrinogen from 340,000 daltons to 334,000 daltons, but the process exposes the necessary polymerization sites to allow formation of aggregated and crosslinked fibrin clots. Jackson, Ann. Rev. Biochem 49: 765-811 (1980); See Furie et al ., Cell 53: 505-518 (1998).

Recently, biological adhesives containing fibrinogen, thrombin, and other components have been developed that produce fibrin clots, similar to the final stage of natural coagulation. In fibrin- or tissue-sealing agents, biological sealants, fibrin- or tissue-glue, biological binders or equivalents thereof (collectively referred to herein as "fibrin sealers"), Tests on these materials show a direct relationship between kidney strength and final fibrinogen concentration (Japanese Patent Unexamined Published Application, Kokai No. Sho61-293443). Thus, the availability of concentrated fibrinogen is important in the manufacture of conventional fibrin sealants.

Fibrinogen-based tissue binders are described, for example, in US Pat. No. 6,117,425 (Mapi et al.). Such preparations also contain fibrinogen and factor XIII, as well as additional proteins such as fibronectin and albumin, and optionally antibiotics, growth factors, and equivalents thereof. Essential catalyst (thrombin-mediated) activity may be derived from the host tissue (wound surface) to be applied or may be added to the tissue binder during the application process in the form of thrombin and Ca ⁺⁺ ion-containing solutions or powders. . The fibrin sealant can be used to seal and / or seal-support bonds of human or animal tissue or organ parts for wound closure, promoting hemostasis and wound healing, coated prosthetic devices, and other therapeutic and non-therapeutic applications. Has been used for

The fibrinogen component of the fibrin sealant is often derived from pooled blood plasma, which is a waste in the manufacture of factor XIII. Fibrinogen can be concentrated from plasma via cryoprecipitation or precipitation via conventional methods using various reactants such as polyethylene glycol, ether, ethanol, ammonium sulfate, glycine and the like. Fibrin sealants are described, for example, in Brennan, Blood Review 5: 240-244 (1991); Gibble et al ., Transfusion 30: 741-747 (1990); Matras, J. Oral Maxillofac. Surg. 43: 605-611 (1985); Lerner et al., J. Surg. Res. 48: 165-181 (1990) et al.

From a manufacturing point of view, according to US Pat. No. 5,290,552, the initial surgical binder formulation essentially contained a high fibrinogen content (about 8-10%), which lyophilized was very difficult to manufacture. The cold precipitation method is relatively unstable, and storage at −20 ° C. is essential until use. The method for improving the stability of the cold precipitation method comprises adding a plasminogen activator or an inhibitor of albumin.

At sufficiently high fibrinogen concentrations, the sample provides effective hemostasis, good adhesion strength and / or wound closure, and complete resorption of the binder, which bonds effective hemostasis, wound and / or tissue portions during the wound healing process. For optimal adhesion, an easy to use binder solution of about 15-60 mg / ml concentration fibrinogen is required (MacPhee, personal communicaition, 1995).

Tissue binders are sold in the form of cold-freezing solutions or in lyophilized state. This is due to the fact that high concentration fibrinogen in liquid solutions is known to be very unstable (http: www.tissuesealing.com/us/pruducts/biological/monograph.cfm), ie they have solidified naturally. As a result, commercially available freeze-dried and / or cold-frozen fibrinogen concentrates, such as Tissucol, may be liquefied prior to use, ie, slowly thawed (“dissolved”) or in their lyophilized form. Should be returned to However, the liquefaction process can result in injured patients in life-threatening situations with considerable effort and time delay before using the product.

The “liquid temperature” of the cold-freeze concentrate, for example, the point of change from liquid phase to the liquid phase, is such that slowly increasing the temperature of the solution to at least 25 ° C., more preferably at least 37 ° C., through considerable stirring for up to 30 to 60 minutes. It is necessary (http: www.tissuesealing.com/us/pruducts/biological/monograph.cfm). As a result, reinstatement of conventional fibrinogen samples necessitated the use of a water bath or other heating device (typically 37 ° C.) capable of converting the cold-freezing material into a ready-to-use solution in the shortest possible time. However, heat exchange is usually very difficult because, for example, a double coated package is required to maintain the product's sterile state over a difficult and cumbersome thawing process. For example, a cold-freeze fibrin sealant made from a pre-filled, ready-to-use, sterile disposable syringe should be double sealed with a plastic film for sterility.

The transition from the cold-frozen solid phase to the liquid phase does not occur abruptly but must go through a temperature increase step that passes through the gelatin and viscous transition. According to at least one test, the sample is not marked as 'liquid' until a test tube is patted to form a uniform liquid phase, ie the sample does not form visible expansion as soon as it is fluid. Thus, testing of the product to determine when it becomes ready to use a uniform "liquid" immediately adds an additional time-consuming step before using the stored conventional fibrinogen sample. In addition, it is clear that the degree of uncertainty and likelihood of mistakes by the user can affect the usability and effectiveness of the fibrinogen product.

The preparation time of lyophilized fibrinogen also causes a significant delay before product use, which can be a substantial problem with recently used fibrinogen-based hemostatic agents. Accordingly, considerable efforts have been made to improve the solubility of lyophilized fibrinogen samples. For example, it was essential for manufacturers to use magnetic stirrers added to protein vials to provide significant agitation during heating. This allows for faster dissolution times than can be obtained for the same product without significant mixing, but requires a preparation time of 30 to 60 minutes to obtain ready-to-use fibrinogen.

Solubility of conventional fibrinogen samples is often further reduced with the implementation of virus inactivation methods. For example, EP 159,311 preferably performs a heat treatment method on the lyophilized material.

It is known that restitution of lyophilisates can be improved through the addition of certain additives. Thus, for example, EP 345,246 describes the preparation of lyophilized fibrinogen which further contains not only fibrinogen, but also at least one biologically acceptable additive (tenside). The addition of the tenside improves the rate of dissolution at certain temperatures rather than the solubility of the fibrinogen itself, resulting in improved wetting of the lyophilisate with the solution. Therefore, the sample must be returned to ambient temperature above 25 ° C., generally 37 ° C.

In order to overcome the need for lyophilization or cold-frozen fibrinogen products, in particular the need to reinstate or liquefy concentrated samples before use, fibrinogen samples that dissolve at room temperature have been introduced. But. This conventional product was cytotoxic (Beriplast, Biocol, Bolheal HG-4).

U. S. Patent No. 5,962, 405 provides a process for the preparation of stable storage lyophilized or low temperature frozen liquid fibrinogen, preferably reconstituted with fibrinogen and / or tissue binders that are readily available without the use of additional methods such as heating and / or stirring devices. A ready-to-use tissue binder solution is prepared which can be returned and liquefied and has a fibrinogen concentration of at least 70 mg / ml. However, the method of preparation comprises fibrinogen and at least one additional material which can improve its solubility and / or lower the liquefaction temperature and reduce the viscosity of the tissue binder solution available immediately at room temperature. The solubility enhancing substance is selected from a substance consisting of benzene, pyridine, piperidine, pyrimidine, morpholine, pyrrole, imidazole, pyrazole, furan, thiazole, purine compound or vitamin, nucleobase, nucleoside or nucleotide. One or more are selected and relatively high proportions of substance / fibrinogen are recommended, but added at a rate of 0.03 to 1.4 mmol per gram of fibrinogen. Additional proteins, adjuvants and additives may also be present. However, because of the low liquefaction temperature, the liquefaction of the low-freezing, concentrated fibrinogen solution of the '405 patent is preferably possible at ambient temperatures of 20 to 23 ° C. (room temperature), as opposed to the conventional warm conditions of 37 ° C. Nevertheless, the method still requires storage under cold-freezing conditions (temperature held below -15 ° C. at −25 ° C.) and the method still takes up to 15 minutes to liquefy.

In another solution for premature coagulation of fibrinogen solution for use as a tissue sealant sample, US Pat. No. 5,985,315 stably includes fibrinogen added with at least one activated coagulation factor whose activity is not dependent on calcium ions. Provided are biologically pre-activated binders. The preactivated binder is stable in aqueous solution, ie, the solution does not spontaneously solidify for at least one hour at a temperature of 20 ° C., but it can be solidified in about 5 minutes simply by adding calcium ions. No additional active agent is required. Thus, the final biological binder does not require the addition of thrombin or prethrombin to coagulate. Unfortunately, however, the slow coagulation time is impractical for use in fluid or oscillating wound types. to be.

Thus, from a medical point of view, a readily available, readily available, biological, tissue binder is essential, especially in surgical emergencies. In addition, it is essential to make ready-to-use fibrin sealant solutions that can minimize the burden on the assistant with as little manipulation as possible. The manufacture of fibrin sealants requires stored fibrinogen components, but at present the fibrinogen can negatively alter the efficiency of lyophilizate, cryo-freeze concentration, or fibrinogen-based tissue binders or safe usability for patients or individuals. Only available in admixture with other ingredients. Accordingly, there is a need for a ready-to-use fibrinogen solution that is stable at high concentrations, is available in fluid form, and has ease of operation in the preparation of tissue binders quickly and easily.

Summary of the Invention

The present invention includes a method of stably storing a readily available biologically suitable mammalian fibrinogen that remains fluid and that can be quickly and easily manipulated with a tissue binder despite high concentrations. Also provided is a sterilized, storage stable, water-soluble fibrinogen product, which is readily available in liquid form, and spontaneously coagulates (ie, forms in the absence of an active agent such as thrombin / Ca ^++). ) And retains biological activity (ie, the ability to rapidly form fibrin clots when exposed to and mixed with thrombin and Ca ⁺⁺ ). The storage concentrated, readily available biologically suitable mammalian fibrinogen of the present invention is completely soluble and the solution is water soluble and its stability is pH and temperature dependent. The product can freeze, thaw, refreeze and re-freeze without affecting the clotting properties of the composition. Examples of mammalian fibrinogen include bovine fibrinogen, but the present invention is not limited thereto, and any other mammalian fibrinogen is possible.

The method of the present invention provides a stable, concentrated, readily available biologically suitable mammalian fibrinogen solution, which stability is maintained for at least one day to one year or more from the date of manufacture.

According to a preferred method, the present invention is prepared freshly, or freshly separated and purified from plasma or any frozen sample, and kept sterile in a suitable container or refrigerator (about 4 ° C.) at room temperature, pH The step of maintaining at a pH in the range of 6.5 to 8.2 provides a ready-to-use fibrinogen solution. Stability is maintained for at least one year. It further provides a ready-to-use, sterile, stable, water-soluble fibrinogen solution stored according to the method of the present invention.

According to another preferred method, the present invention provides a step of enhancing storage stability by adding a protease inhibitor to the readily available fibrinogen solution described above. Accordingly, the present invention provides a method of preparing fibrinogen solution freshly under sterile conditions, or freshly separating and purifying fibrinogen solution from plasma; Adding an effective amount of a protease to the fibrinogen solution to prevent protein hydrolysis of the fibrinogen sample; And (i) constant temperature in the range of about 4 to 23 ° C., wherein the fibrinogen solution is liquid; providing a method of safely storing a mammalian fibrinogen in a ready-to-use aqueous solution comprising (ii) a pH level in the range of pH 6.31 to 8.1, and (iii) storage under conditions in which the biocompatibility and biological activity of the fibrinogen is maintained. do. Stability is maintained for at least one year. It further provides a ready-to-use, sterile, stable, water-soluble fibrinogen solution stored according to the method.

In certain embodiments, other additives or ingredients are also added to the above-mentioned storage stable, readily available fibrinogen solution to enhance the efficiency of the final fibrinogen in subsequent applications or in products or materials prepared therefrom. It further provides a ready-to-use, sterile, stable, water-soluble fibrinogen solution stored according to the other methods above.

The ready-to-use, concentrated mammalian fibrinogen solution prepared and stored as described above may be used by neutralization without additional steps or processes in the preparation of biological tissue binders or sealants, including the preparation of fibrin sealants on the fly, eg For example, for wound healing, coagulation, fibrosis, suppression of postoperative or postoperative sequelae, coated angioplasty, or infusion purposes, as well as other additional or in vivo in vivo or in vitro. Pharmaceutical or cosmetic uses including additional therapeutic or non-therapeutic applications.

Other objects, advantages and novel features of the present invention will be further described in the following detailed description, examples, and drawings, and the contents through the test or implementation of the present invention will be apparent to those skilled in the art.

The present invention relates to a process for producing concentrated fibrinogen that is stable in storage and to preventing blood loss and to use it in wound healing and many other therapeutic and non-therapeutic applications.

The present invention claims priority to US Provisional Application No. 60 / 326,963, filed October 3, 2001, and is incorporated into the present invention.

The above summary and the following detailed description of the invention are intended to be further understood through the accompanying drawings. For the purpose of illustrating the invention, the drawings show preferred embodiments of the invention. However, the present invention is not limited to the specific contents shown in the following drawings.

1A and 1B are photographs showing non-reducing (FIG. 1A) and reducing SDS PAGE (FIG. 1B) using bovine fibrinogen samples after storage for 44 days at room temperature. The lanes in each of the two gels are identical. 1 = standard MW; 2 = bovine fibrinogen control; 3 = sample under histidine buffer pH 7.24; 4 = sample under pH 9.31 buffer; 5 = sample under pH 9.05 carbonate buffer; 6 = sample under pH 9.86 carbonate buffer; 7 = bovine fibrinogen control.

The present invention provides a method for the stable storage of readily available fibrinogen which, despite its concentration, can be maintained in a fluid state and which makes tissue binder preparation easy and fast to proceed. The present invention also provides a storage stable, water soluble fibrinogen product made using the method.

The ready-to-use, water-soluble fibrinogen solution of the present invention remains stable in the liquid state even after a period of several days, is free of spontaneous coagulation (ie, forming a coagulum under an active agent sub such as thrombin / Ca ++), and biologically active (ie, Strong mixing when exposed to thrombin and Ca ⁺⁺ ) is "storage-stable" when the ability to rapidly form fibrin clots is maintained. The method of the present invention describes the conditions under which fibrinogen is stored in a ready-to-use, aqueous solution for a substantial period of time and that activity and stability are maintained (storage-stable).

"Activity" as used for storage-stable fibrinogen solutions means the "biological activity" of the protein, said "biological activity" as described above, in part or in part, such as the ability to rapidly form fibrin clots, as described above. Or in vivo, means one or more activities associated with fibrinogen. Methods of assessing biological activity are well known in the art.

In the present invention, unless otherwise defined, all technical and scientific terms used have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

The storage method of the present invention applies to any fibrinogen sample that has been isolated and purified into plasma, or recombinantly prepared, or freshly isolated or freshly prepared from a lyophilized or cold-frozen sample. The method of the present invention is a time for freezing or cold-freezing the fibrinogen sample as long as the biological activity of the freshly prepared fibrinogen solution is the same as the equivalent sample isolated and purified from plasma and spontaneous clotting is not induced in the solution. Applicable regardless of length

One preferred embodiment of the present invention is directed to the preparation of a final, concentrated fibrinogen having crude fibrinogen product, or greater than 90% protein purity and more than 95% clotable protein, during the preparation process, or any fibrinogen at both concentrations above Applicable. For example, in the examples below, bovine fibrinogen production has a protein purity of 61% and a clotable protein of 97 < >, whereas in other examples performed by the inventors using human fibrinogen (not shown). The preparation has 53% protein purity and 95% clotable protein. Nevertheless, the method of the present invention is applicable to both of the above embodiments.

In a preferred and representative embodiment of the present invention, the storage method was applied to concentrated bovine fibrinogen samples. Although highly concentrated, the storage-stable fibrinogen sample of the present invention remains dissolved in an aqueous solution so that the fibrinogen is particularly suitable for use in the preparation of additional or non-additive, ready-to-use biological tissue binders. The fibrinogen is used in the preparation of ready-to-use tissue binder samples, at a concentration of 10 to 85 mg / ml, more preferably at a concentration of 15 to 75 mg / ml, more preferably at a concentration of 30 to 70 mg / ml, And most preferably at a concentration of 40 to 65 mg / ml.

In addition, the concentration of the fibrinogen, or fibrinogen-containing protein in the storage-stable aqueous solution of the present invention is generally in the range of 2 to 10 w / v%, preferably 4 to 7 w / v%. The concentration of fibrinogen is determined by protein absorbance measurement at 280 nm (the absorbance of 1% fibrinogen solution is 14).

The storage-stable fibrinogen of the present invention is completely dissolved in an aqueous solution, that is, a solution of water type. The optimum temperature and pH of the sample have been identified through the present invention and can be readily determined using one of the common methods well known to those skilled in the art. However, water-soluble gels can also be used in the present invention, in which such materials completely dissolve the fibrinogen contained therein, and the sample is sufficiently fluid and ready for immediate biological tissue binder preparation or the present invention. Other applications involving storage in accordance with may be used if possible. The core of the present invention is that the fibrinogen solution is stably stored in a usable fluid state; It is not stored in a dry freeze sample or in a frozen state.

In one preferred embodiment of the present invention, even though the viscosity of the fibrinogen solution is typically greater than water, the fresh fibrinogen solution is a free flowing liquid that can easily move along the inverted test tube. Biologically active storage samples (ie, coagulation in the presence of thrombin and Ca ions) essentially have the same physical characteristics as fresh samples. This type of clot produces a controlled clot formed using active fibrinogen when a tissue binder is made and used. The purpose of this type of clot is referred to simply herein as "fibrin clot" in the present invention to distinguish it from "voluntary clot", the latter can occur in concentrated fibrinogen, which is unstable even in the absence of thrombin or other active agents.

However, the term suggests the preferred use of the stored fibrinogen solution with the activity of a stably stored fibrinogen solution, which is demonstrated through the rapid formation of fibrin clots when the same amount of fibrinogen is mixed with thrombin / Ca ⁺⁺ . It is used only for the purpose of distinguishing it from spontaneous clot which means instability of the conventional fibrinogen solution. Conventionally, freshly prepared fibrinogen aqueous solutions have been very unstable, and the storage of fibrinogen in liquid form, which is readily available after one or two days of storage using conventional methods, has been impractical due to the tendency to spontaneously coagulate upon storage.

Spontaneous clotting (eg, without exposure to active agents such as thrombin and Ca ions) was recognized as an increase in viscosity, resulting in a noticeably reduced motility (flow) upon mixing. Often spontaneous clotting occurred in conventional freshly prepared water-soluble fibrinogen solutions in less than one day, often only a few hours. The process is irreversible and renders the fibrinogen useless in such uses as the manufacture of fibrin sealants. The instability makes it impossible to fully predict the length of time that fibrinogen can be stored in a form readily available using recent methods and thus was unreliable.

In a preferred embodiment of the invention, the storage-stable fibrinogen is stored in a polymer, plastic or plastic container, more preferably the plastic container is polypropylene. Glass is not used for fibrinogen or platelet storage because it enhances spontaneous clot formation.

A ready-to-use storage solution of fibrinogen that is present as a fluid (with a viscosity similar to water) and which does not coagulate even with the addition of thrombin and calcium ions is called "thrombin-insensitive". However, the analysis of such thrombin insensitive fibrinogen via SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) shows that the fibrinogen protein is irreversibly degraded into low molecular weight fragments. Thus, samples that no longer contain active fibrinogen are not used in the present invention.

After addition of thrombin / Ca ⁺⁺ to a readily available fibrinogen solution, a rapid increase in viscosity and a decrease in liquid motility are called "gels". In this gel state, the fibrinogen solution no longer flows freely and must be allowed to move through stirring. Although this measure is subjective, the estimated variability is only ± 2 seconds.

"Clot" formation is the abrupt solidification of a fibrinogen solution that is unable to flow liquid phase from solid material through stirring. The antifreeze material is visually opaque white and viscous. Scanned electron micrograph (SEM) photographs of conventional physiological or nonphysiological fibrin clots are described, for example, in Redl et al. Medizinische Welt 36: 769-76 (1985). The clot is generally attached to the wall of the test tube and is not removed even if the tube on the solid surface is severely hit. This measurement is less subjective than gel formation and is slightly larger for slow clot (> 100 seconds) samples, but the estimated uncertainty was only ± 1 second for fast formed samples (8-12 seconds).

The temperature of the solution during storage is not particularly limited as long as the contained fibrinogen remains stable (ie without inert or spontaneous clot). Preferred temperatures for storage of the fibrinogen solution of the invention range from 1 to 25 ° C, more preferably from 4 to 23 ° C. At refrigeration, the optimum temperature is 4 ± 1 ° C. When stored at room temperature, the optimum temperature is in the range of 20-25 ° C., more preferably 22-24 ° C., and most preferably 23 ± 1 ° C.

To assess the effect of clot formation after freezing, samples of fibrinogen solution were frozen and thawed prior to testing, and at least one freeze / thaw cycle was negative for the coagulability of mammalian fibrinogen solution even after storage at 4 ° C. for 5 months before freezing. It was confirmed that there was no effect. Together, the results clearly show that liquid fibrinogen can be easily formulated so that liquid fibrinogen initially freezes to provide at least one year of storage with an additional number of years of storage.

The pH value of the aqueous fibrinogen solution is preferably titrated to about pH 5-8, more preferably pH 6.2-7.5 during storage. The optimal pH for the storage of a particular fibrinogen solution depends on the temperature at which the material is stored, as shown in the table accompanying the Examples below. However, according to the information provided in the present invention, those of ordinary skill in the art know the determinants in which the contained protein remains stable (ie, without inertness or spontaneous clotting), and therefore, based on the planned storage temperature and conditions, The optimal pH can be chosen.

For example, ready-to-use bovine fibrinogen (without protease inhibitors) stored at room temperature (-23 ° C.) is optimally maintained at pH 6.5-9.0, preferably at about pH 6.5- 8.2, to neutralize stored samples and thrombin / When exposed to Ca ⁺⁺ , the ability to form clots quickly is maintained. If the readily available fibrinogen (without protease) is stored in refrigeration (˜4 ° C.), the optimum pH is also kept optimally stored at pH 6.5 to 9.0, preferably about pH 6.5 to 8.2, more preferably pH 6.5 to 7.07 The ability to quickly form clots is maintained when the sample is neutralized and exposed to thrombin / Ca ⁺⁺ (see Table 2).

The pH of the storage-stable fibrinogen solution is determined via storage buffer. For example, in the following examples, bovine fibrinogen solution (50 mg protein / ml) was freshly prepared with one of the following 0.1 M buffers: histidine, pH 7.24; Glycine pH 9.31; Or carbonate, pH 9.05 or pH 9.86.

In a preferred embodiment of the present invention, other physiologically acceptable buffers that can be used to prepare storage-stable fibrinogen are known, but the final pH of the fibrinogen solution is maintained in a defined range so that activity is maintained and spontaneous clotting is achieved. Storage-stable bovine fibrinogen solution is prepared in histidine buffer as long as it is free.

Commercially available fibrinogen, available recently, contains the salts used in the separation and purification processes. As mentioned in the Examples, the presence of salts, including sodium citrate and sodium chloride, but the remainder of such fibrinogen purification, does not affect the storage-stability of the final sample. The object of the present invention is to produce a storage-stable, ready-to-use fibrinogen solution that retains the properties of comparable, freshly prepared fibrinogen solutions, so the effect of the fibrinogen purification process is the same. Nevertheless, high concentrations of citric acid and / or sodium can affect the coagulation of stored fibrinogen samples. Thus, even if the same salt or other chelate compound is present in a freshly prepared solution, the present invention is effective and storage stable as long as the activity is maintained during storage and spontaneous clotting is not induced by the salt or chelate compound. Retains the properties and activity of a freshly prepared solution that is comparable.

For the purposes of the following examples, sodium azide (0.025%) was added to each sample as an antimicrobial agent. Although the antimicrobial agent may induce spontaneous clot to some extent, this effect did not appear. In a preferred embodiment of the present invention, no antimicrobial agent is added and sterility is preserved using the prior art. However, in another embodiment, some degree of antibacterial agent was added to prevent microbial contamination of the fibrinogen solution stored for a long time. Physiological antibacterial agents are acceptable for the purposes of the present invention, as long as the activity of the fibrinogen solution is maintained during storage and no spontaneous clot is induced.

The storage-stable fibrinogen solution of the present invention may be added to act as a carrier carrier, such as growth factors, drugs or other compounds or mixtures thereof, as long as the activity of the fibrinogen solution is maintained during the interpolation period and no spontaneous clot is induced as described above. May be added. For example, if growth factors are added to the fibrinogen sample, the fibrinogen that is readily available when used in the manufacture of fibrin sealants or tissue conjugates may accelerate, promote, and improve wound healing, tissue regeneration. Such additional samples may also include additional components such as, for example, drugs, antibodies, anticoagulants and other compounds, which compounds (1) increase and promote the biological activity of growth factors or other additives or components. Or mediate; (2) reduce the activity of one or more additives or components of the fibrinogen added with a growth factor or fibrin sealant or tissue binder prepared therefrom, wherein the activity is an activity that inhibits or destroys the growth factor in the sample; (3) sustained arrival of additives or components from samples such as fibrin sealants or tissue binders prepared from the readily available fibrinogen solution of the present invention; (4) It has another preferable characteristic. Such additives or supplements include mutants, derivatives, truncated forms or other modified forms, which have a biological activity similar to the compound or composition derived.

One or more additives or components may be added or supplied simultaneously to the storage-stable fibrinogen solution of the present invention. The concentration of the additive and / or component may vary in the fibrinogen solution, depending on the purpose, and the concentration should be sufficient to accomplish the intended purpose of the component and / or composition. The amount of adjuvant added can be empirically determined by those skilled in the art to test various concentrations and to be selected effectively for the intended purpose and field. Dyestuffs, tracers, labels, and equivalents can also be added, for example to confirm the subsequent arrival of the substance to which the fibrinogen has been added.

In a preferred embodiment of the invention, a protease inhibitor (PI), for example, but not limited to, aprotinin (eg, 5 μg / ml final concentration) or PPACK (eg, 25 uM final concentration) Was added to the water-soluble fibrinogen solution stored in an effective amount. Other protease inhibitors (PIs) are known in the art and can replace the aprotinins and PPACKs described in Example 2. In particular, aprotinin is a commercially available Tisseal product. An “effective amount” of protease inhibitors refers to the amount of PI that can prevent proteolysis of the fibrinogen sample. The amount varies depending on the PI or mixture of PIs used, but can be readily determined by those skilled in the art. However, even though the fibrinogen solution stored in the presence of PI remained stable for long periods of time, the PI effect decreased over time.

For example, the addition of PI to storage-stable bovine fibrinogen samples may inhibit undesirable spontaneous clot formation of proteins stored at ˜4 ° C. for long periods of time, but the addition of PI may, for example, result in rapid fibrinogen at 149 days. It was not effective for use in producing / thrombin products (fibrin clots). However, rapid fibrinogen / thrombin clot formation was seen in storage-stable, bovine fibrinogen solution samples maintained at room temperature (-23 ° C.) and pH 6.3 to 7.07 for at least 149 days.

As shown in Tables 2 and 3, "inhibition" is equivalent to "prevention", in particular, PIs are initially active under the conditions disclosed in the present invention (i.e., clotting is inhibited / prevented). The inhibitory effect is reduced and the activity of PI is reduced after substantially stopping. The rate of decrease of PI activity in the fibrinogen solution was pH and temperature dependent.

The examples carried out in the present invention, through repeated observation and testing, are stable (active and at least 97 days at pH 6.5 to 9.0 when the fibrinogen solution of the present invention is stored at room temperature (-23 ° C.) under preferred conditions. No spontaneous clot), stable for at least 149 days at pH 6.5-8.1 in the presence of PI when stored at ˜4 ° C., but only 7 days in the absence of PI. Thus, the fibrinogen solution of a preferred embodiment of the present invention comprising PI with fibrinogen is stable for several years at room temperature and for several months in the absence of PI.

In view of the proven stability of the bovine fibrinogen solution, fibrinogen is maintained for a very long time compared to a cold freeze or lyophilized sample of concentrated protein maintained without substantial loss of activity (i.e., fibrinogen / thrombin fibrin clot still forms rapidly upon mixing). It can be seen to be stable for many years after the initial storage of the product. Thus, “long term storage” is for at least 3 days, preferably for at least 3 weeks, more preferably at least 13 weeks, more preferably at least 149 days, more preferably at least 1 year, and most preferably 1 Storage of fibrinogen solution in a form readily available under the conditions disclosed herein, without substantial loss of protein activity for more than a year. In addition, the term is meant to further include a frozen storage period in addition to the storage of the product in a usable form, in addition to the additional years of storage of the product.

The present invention relates to all fibrinogen samples, but the method of the present invention relates to the stable storage of readily available, water-soluble fibrinogen solutions in all mammals. Although the fibrinogen of bovine was described in the Examples, the present invention is not limited thereto. There was no species suitability associated with the use of storage fibrinogen for other mammals. For example, fibrinogen in bovine subjects can be used after storage in aqueous solution, for example, to produce biologically suitable binder samples that can be used in any kind of mammal.

Fibrinogen, a plasma protein, is often accompanied by the risk of contamination by more specifically blood-borne pathogens such as hepatitis virus or HIV, which can contaminate the plasma protein. Thus, those skilled in the art immediately produce fibrinogen to remove the infectious material as much as possible. General techniques for achieving the above objectives include, but are not limited to, ultrafiltration, pasteurization (heating), solvent detergent treatment, radiation exposure, and ultraviolet treatment. Virus inactivation by the high heat or steam method is impractical in biologically active protein solutions comprising the fibrinogen solution of the present invention, wherein nanofiltration is an optional treatment prior to placing the fibrinogen solution of the present invention in a sterile storage container.

Although fibrinogen is thermally unstable and inactivated during conventional liquid heating processes, it is common for processes to heat fibrinogen, for example, to inactivate possible contaminating viruses such as hepatitis or HIV without fibrinogen itself. It has been developed. U. S. Patent No. 5,116, 950 (Miyano et al ., Issued May 26, 1992) includes heating a fibrinogen-containing aqueous solution until the virus contaminated fibrinogen is inactivated, at least in the presence of sugars, amino acids and magnesium salts. Provide a heating method.

In one preferred embodiment of the invention, the aqueous fibrinogen solution is heated, preferably more purified and proceeds with conventional methods such as dialysis, sterilization or filtration. In addition, various washing steps are applied to remove stabilized additives by conventional methods known in the art.

The fibrinogen solution of the present invention is very ideal for forming physiological fibril structures when exposed to the activator solution, and fibrin clots are formed quickly. This resulted in a fibrinogen solution stored in excess of 3 to 6 mM CaCl ₂ in an equal amount of thrombin / CaCl ₂ solution (eg 2.5 units / mg fibrinogen (100 units / ml), thrombin and citric acid or other chelate compounds added to the solution). It was demonstrated through mixing with), and is shown in the following examples. If clot is formed by thrombin activity in freshly prepared or freshly isolated and purified bovine fibrinogen at fresh physiological conditions, ie an ionic strength of about 0.15 and neutral pH, the final coagulation is typical of the physiological fibrin structure demonstrated. It has a spatially branched fibril structure.

Fibrinogen and thrombin concentrations depend on clot formation time, clot strength, clot cohesion and thus hemostasis.

In addition, the fibrinogen sample and / or fibrinogen-based tissue binder added according to the present invention did not have a cytotoxic effect when used as a tissue binder, ie, "biologically suitable" means that the cells are well adapted, This provides an ideal prerequisite for good and excellent wound healing. This was demonstrated by diluting the tissue binder with the same amount of mid-isotonic solution or isotonic sodium chloride solution and adding it to the growth medium of fibroblasts. No deleterious effects were observed on fibroblasts (Redl et al. , 1985).

Thus, the storage-stable, ready-to-use fibrinogen solution of the present invention is prepared with all the requirements of tissue binders, namely biocompatibility, viral safety and high bonding strength, and is also simple and fast from the readily available fibrinogen product. There is an advantage to manufacture. Tissue binders prepared from the storage stable fibrinogen of the present invention include injection purposes such as delivery of antibiotics, anti-tumor drugs, anesthetics, and equivalents thereof, which are biologically prepared and additional or non-additive tissue binders are used. For example, pharmaceutical or plastic use; For wound healing, coagulation, and fibrinemia; For suppressing sequelae after or after surgery; For coating prosthetics; It can be used in any conventional field, such as for wound closure to be dressed and for safe and persistent hemostasis, i.e. for patient fluid and / or air leaks and the like.

The invention is further illustrated by the following examples. However, the following examples are provided to those skilled in the art for illustrative purposes, and are not intended to be limiting. Moreover, the following examples are not to be construed as limiting the scope of the appended claims. Accordingly, the present invention is not limited to the following examples and includes all modifications that may be demonstrated by the results provided in the present invention.

In order to evaluate the storage-stability of the fibrinogen solution of the present invention, the stability, solubility and coagulation activity of the fibrinogen solution was evaluated for various storage conditions with additives such as different buffer solutions (pH values), temperature, and protease inhibitors. Bovine fibrinogen, bovine thrombin, aprotinin, buffer solution, calcium chloride, sodium hydroxide and hydrochloric acid were purchased from Sigma Chemical, St. Louis, Missouri. PPACK was purchased from Calbiochem, San Diego, California. It has been demonstrated that bovine fibrinogen contains 61% protein (97% clotable) and 39% salt.

Standard irradiation grade fibrinogen contains the salt used during the separation and purification process. The fibrinogen includes sodium citrate and sodium chloride. Thus, a 40 mg / ml fibrinogen solution contains, for example, 54 mM sodium citrate and 419 mM sodium chloride with fibrinogen. In addition, sodium azide (0.025%) was added to each sample as an antimicrobial agent.

Coagulation analysis was carried out in a general manner according to Casper (Proc. Symposium on Recent Advances in Hemophilia Care, Los Angeles, CA April 13015, 1989 (in Liss, New York, 1990)) A portion of each fibrinogen sample (100). [Mu] l) was placed in a 4 ml polypropylene test tube, each sample was neutralized (pH7.0-7.3) with 0.1 M sodium hydroxide, 0.2 M histidine buffer (pH 6.0), or 0.1 M hydrochloric acid (greater than Thrombin was prepared at 200 units / ml with 1 M calcium chloride (3-6 mM excess calcium over sodium citrate in the fibrinogen sample). Dilution to 2) resulted in a final thrombin concentration of 100 units / ml (2.5 units of trobin per mg fibrinogen) All samples were analyzed at room temperature (23 ± 2 ° C.).

Coagulation was determined by adding 100 μl of thrombin to the fibrinogen sample (100 μl) and measuring the time of reaction that occurred when the mixture was mixed vigorously. The time when the solution became a viscous gel (the liquid to be mixed very slow) to a solid form (the point at which all liquids ceased on mixing) was recorded. The time to solidify coagulation was usually twice the gel formation time.

Example 1

Stability of water-soluble bovine fibrinogen stored at room temperature, pH 7-10

In order to evaluate the ability to store the fibrinogen of the present invention for a long time at room temperature, the stability, solubility, and coagulation activity of the fibrinogen solution were evaluated after storage for at least 149 days (21 weeks) at a constant temperature of 20 to 25 ° C. Bovine fibrinogen solution (50 mg protein / ml) was freshly prepared for one day in one of the following 0.1 M buffers: histidine, pH 7.24; Glycine pH 9.31; Or carbonate, pH 9.05 or pH 9.86.

The transparency and spontaneous clot of the solution were investigated. Passive coagulation assays were performed at 25 ° C. by neutralizing the solution to pH 7.0-7.5 and adding thrombin (125 units / mg fibrinogen) and 3-5 mM excess CaCl ₂ over the citric acid of the fibrinogen solution. The sample was mixed vigorously and the time required to form a clot was measured and recorded as described above.

Table 1 shows the results of coagulation of bovine fibrinogen in histidine buffer at pH 7.24 stored at room temperature (˜23 ° C.). In all samples, for 1 to 149 days, the fibrinogen solution remained clear and did not form a clot until thrombin was added.

Work Clot Time (sec) pH 7.24 pH 9.05 pH 9.31 pH 9.86 One NT NT NT NT 3 9 8 8 27 36 10 > 300 > 300 > 300 72 9.5 > 300 > 300 > 300 149 > 300 NT NT NT

NT = not tested.

Protein integrity of the fibrinogen formulations was evaluated on day 44 via sodium dodecyl sulfate (SDS) -polyacrylamide gel electrophoresis (SDS-PAGE) (standard SDS described in Laemmli, Nature 227: 680-685 (1970), etc.). -PAGE condition). The SDS-PAGE analysis showed that the bovine fibrinogen sample (lane 3 in FIG. 1) stored at pH 7.24 was identical to the bovine fibrinogen (BFG) control (lanes 2 and 7 in FIG. 1) freshly prepared in non-reducing and reducing gels. Indicates moving at speed. In contrast, samples stored at high pH (lanes 4, 5, and 6 in FIG. 1) were degraded and / or aggregated. Degradation was greatest at pH 9.05 to 9.31 (lanes 4 and 5 in FIG. 1), with less degradation and more coagulation (or coagulation) in the pH 9.86 sample.

Concentrating on a bovine fibrinogen solution at pH 7.24, the sample remained clear and non-coagulated even after 149 days storage at room temperature. However, in a single coagulation assay, the pH 7.24 sample did not coagulate upon thrombin addition. pH 7.24 The pH of the sample was determined to be 6.98 after thrombin addition. Neutral pH is optimal for thrombin. Nevertheless, the samples lost clotting ability between 73 and 147 days.

Thus, it was concluded that bovine fibrinogen prepared in aqueous solution in histidine buffer at pH 7.24 was stable upon storage at room temperature for 10 weeks or more. However, it did not clot at 21 weeks.

Example 2

Stability of Stored Water-soluble Fibrinogen Solution at Two Temperatures with or Without Protease Inhibitor

To further assess the ability to store long-term water-soluble fibrinogen solutions, the stability, solubility, and coagulation activity of the two bovine fibrinogen solutions ranged from five pH (pH 6.50 to pH 9.87), presence of protease inhibitors (PI), and room temperature. Evaluation after storage for at least 149 days (more than 21 weeks) at (-23 ° C.) and refrigerated (˜4 ° C.) temperatures. Dual samples of bovine fibrin (39 mg protein / ml) were freshly prepared in one day stored in one of the following 0.1 M buffers: histidine, pH 6.0 or 7.2; Tris pH 8.16; Glycine pH 9.3; Or carbonate, pH 9.1 or pH 9.9. Protease Detergent: PPACK (25 uM final concentration) and Aprotinin (5 μg / ml final concentration) were added to one of the two samples prior to storage.

In order to evaluate the clotting ability, it was neutralized according to the protocol described above, and the clotting was tested as described in Example 1.

The results of coagulation of bovine fibrinogen according to the conditions are shown in Table 2.

Clotting time of bovine fibrinogen stored without protease inhibitors at 23 ° C. and 4 ° C. Work Temperature, ℃ Clot Time (sec) pH 6.5 pH 7.36 pH 8.2 pH 9.04 pH 9.87 4 23 12 13 15 12 210 4 10 9 15 10 Clot 7 23 10 10 11 11 240 4 11 10 10 10 Clot 22 23 9 10 10 > 300 > 300 4 Partial clot Partial clot Clot Clot Clot 97 23 10 100 > 300 > 300 Clot 4 Clot Clot Clot Clot Clot 149 23 Clot > 300 > 300 > 300 > 300 4 Clot Clot Clot Clot Clot

Note: "Clot" means spontaneous clot in the absence of thrombin.

On day 22, the samples at 4 ° C. all spontaneously solidified. In contrast, when measured at day 97, bovine fibrinogen samples stored at room temperature were nearly clear except for the highest pH samples.

Clotting time of bovine fibrinogen stored in the presence of protease inhibitors at 23 ° C. and 4 ° C. Work Temperature, ℃ Clot Time (sec) pH 6.31 pH 7.07 pH 8.10 pH 9.09 pH 9.80 4 23 40 30 120 26 300 4 > 300 > 300 > 300 180 60 7 23 15 25 60 20 > 300 4 > 300 > 300 40 60 22 22 23 15 12 20 65 > 300 4 > 300 100 95 15 15 97 23 30 28 > 300 > 300 > 300 4 18 24.5 21 NT 130 149 23 180 125 > 300 > 300 > 300 4 25 15 15 Clot > 300

NT = no measurement. "Clot" means spontaneous clot in the absence of thrombin.

Samples containing PI (PPACK or Aprotinin) evaluated after storage at -23 ° C and -4 ° C showed pH dependent results. The reduced clotting ability was due to the ability of residual PI in the fibrinogen solution to inhibit added thrombin. Thus, shorter storage at ˜4 ° C. (4 to 22 days) resulted in an effective inhibition of thrombin-dependent clotting, ie the activity of thrombin after the addition of thrombin is left by the residual PI activity remaining in solution. The sample did not clump as it was inhibited.

However, since PI components decrease with time, their activity gradually decreases. After a longer storage period (22-149 days), PI activity was reduced, and therefore the addition of thrombin resulted in the coagulation of the fibrinogen sample. In addition, the reaction was pH-dependent.

As a result, after storage for at least 149 days, the optimal conditions for storing bovine fibrinogen in aqueous solution were 4 ° C. with a pH ranging from 6.31 to 7.07 and room temperature, or between 6.31 and 8.10 in the presence of a protease inhibitor.

Each and every patent, patent application, and publication cited herein is incorporated by reference.

The present invention has been described in terms of preferred embodiments and many specific examples have been described for purposes of explanation. It will be apparent to those skilled in the art that the present invention can be applied to various modifications and additional embodiments, and that the details described herein can be significantly changed without departing from the spirit and scope of the invention. Modifications, equivalents, and variations of the above and additional embodiments are also within the scope of the appended claims.

Claims (30)

A storage stable, concentrated, readily available biologically suitable mammalian fibrinogen solution, wherein the stability of the fibrinogen solution is pH and temperature dependent. The fibrinogen solution of claim 1, wherein the fibrinogen is completely dissolved and the solution is water-soluble. The fibrinogen solution according to claim 1 or 2, wherein the stability of the fibrinogen solution is maintained for a storage period of at least 1 day to at least 1 year from the date of manufacture. The fibrinogen solution of claim 1, wherein the fibrinogen solution comprises a pH-adjusted buffer selected from the group consisting of histidine, tris, glycine or carbonate. 5. The fibrinogen solution of claim 1, wherein the solution is buffered to a pH range of pH 6.5 to 8.2 and the storage temperature is maintained under refrigeration at a temperature of about 4 ° C. 6. 6. The fibrinogen solution of claim 5, wherein the storage buffer is histidine. 6. The fibrinogen solution of claim 1, wherein the stability is maintained for at least about 10 weeks. 7. The fibrinogen solution according to any one of claims 1 to 4, wherein the solution is buffered in a pH range of pH 6.5 to 8.2, and the storage temperature is maintained at room temperature. The fibrinogen solution of claim 8, wherein the stability is maintained for at least 7 days. The fibrinogen solution of claim 8, wherein said stability is maintained for at least 22 days. The method of claim 1, wherein the solution is buffered to a pH range of pH 6.31 to 8.1, the storage temperature is maintained at a temperature range of about 4 to 23 ° C., and an effective amount of protease inhibitor is stored. A fibrinogen solution, characterized in that it is added to the fibrinogen solution before to prevent proteolysis of the fibrinogen sample. The fibrinogen solution of claim 11, wherein said stability is maintained for at least 7 days. The fibrinogen solution of claim 11, wherein said storage stability is maintained for at least 22 days. The fibrinogen solution of claim 11, wherein said stability is maintained for at least 97 days. The fibrinogen solution of claim 11, wherein said stability is maintained for at least 149 days. The fibrinogen solution according to any one of claims 1 to 15, wherein the mammalian fibrinogen is a bovine fibrinogen. Preparing a fibrinogen solution freshly separated and purified from a freshly prepared fibrinogen solution or a plasma or frozen fibrinogen sample under sterile conditions; Under sterile conditions, storing the freshly prepared or freshly separated and purified fibrinogen solution, and Maintaining the stored fibrinogen solution under refrigeration temperature, Wherein said fibrinogen solution is liquid, has a pH level in the range of pH 6.5 to 8.2, and is maintained under conditions in which the biocompatibility and biological activity of the fibrinogen is maintained. 18. The fibrinogen solution of claim 17, further comprising maintaining stability for at least one day to one year or more from the date of manufacture. 19. The method of any one of claims 17-18, wherein the refrigeration temperature is maintained at about 4 ° C. 20. The method of any one of claims 17-19, wherein said stability is maintained for at least 7 days. Storing fibrinogen solution prepared from freshly prepared or freshly separated and purified fibrinogen solution or frozen fibrinogen sample under sterile conditions, and Maintaining the stored fibrinogen solution at room temperature; Wherein said fibrinogen solution is liquid, has a pH level in the range of pH 6.5 to 8.2, and is maintained under conditions in which the biocompatibility and biological activity of the fibrinogen is maintained. 22. The fibrinogen solution of claim 21, wherein the method further comprises maintaining stability for at least one day to one year or more from the date of manufacture. The method of claim 22, wherein said stability is maintained for at least 7 days. The method of claim 22, wherein said stability is maintained for at least 22 days. Preparing fibrinogen solution freshly under sterile conditions or freshly separating and purifying fibrinogen solution from plasma or frozen fibrinogen samples; Adding an effective amount of a protease inhibitor to the fibrinogen solution to prevent proteolysis of fibrinogen samples; And The fibrinogen solution is (i) in a constant temperature range of about 4 to 23 ° C., wherein the fibrinogen solution is in liquid phase; safely storing the mammalian fibrinogen in a ready-to-use aqueous solution, comprising (ii) a pH level in the range of 6.31 to 8.1, and (iii) storing the fibrinogen under conditions that maintain its biocompatibility and biological activity. How to. The method of claim 25, wherein the method further comprises maintaining stability for at least one day to one year or more from the date of manufacture. 27. The method of claim 26, wherein said stability is maintained for at least 7 days. 27. The method of claim 26, wherein the stability is maintained for at least 22 days. 27. The method of claim 26, wherein the stability is maintained for at least 97 days. 27. The method of claim 26, wherein the stability is maintained for at least 149 days.